The present invention relates to a DNA sequence which comprises a nucleotide sequence encoding the Hemorrhagic Enteritis Virus, to proteins encoded thereby, to vectors and DNA constructs comprising the said DNA sequence or essential fragments thereof, and to various uses of the DNA sequence and the proteins encoded thereby.
One of the principal diseases which suppress the immune system in turkeys is caused by infection with Hemorrhagic Enteritis Virus (HEV). HEV belongs to the Adenoviridae family. This family consists of serotypes that infect mammals (Mastadenoviridae) and avian (Aviadenoviridae) [Shenk T. Virology, pp. 2111-2148 (1996), B. N. Fields, D. M. Knipe and P. M. Howley (Eds) Lippincott-Raven New York]. HEV, together with marble spleen disease of pheasant and splenomegaly virus of chickens, are classified as type II avian adenovirus (Ad) [Domermuth C. H. and Gross W. B., Diseases of Poultry 8th ed. Pp.511-516, H. J. Barnes, B. W. Calnck, W. B. Reid and Yoder H. W. (Eds) Iowa State University Press (1984)] which is serologically distinct from type I and type III isolated from chickens infected by fowl Ad (FAV) 1-12 and egg drop syndrome (EDS) virus, respectively.
The HEV, as other type II Ad""s, is a non-enveloped DNA virus, with a diameter of about 70-90 nm and an icosahedral symmetry. The genome is linear, double-stranded DNA and with a size estimated to be approximately 25.5 kb [Jucker et al J. Gen. Virol. 77:469-479 (1996)]. Partial sequence (about 4 kb) of the HEV genome has been recently published [Jucker et al. (1996) ibid.], while full sequences of several human Ad""s (types 2, 5, 12 and 40), avian Ad""s (CELO and egg drop syndrome (EDS)) and ovine Ad were published and may be found in EMBL and GenBank data bases. The e organization of all human Ad""s is very similar [Shenk T. (1996) ibid.]. However, in some non-human Adenovirus (Ad) sequences (CELO, Ova and EDS) no similarity was found for various regions of human Ad [Chiocca, S., et. al. J. Virol. 70:2939-3949 (1996); Vrati, S., et. al Virology 220:186-199 (1996); Hess, M. et. al., Virology 238:145-156 (1997), respectively].
HEV replicates in the host cell nucleus and consists of 11 proteins, encoded by its DNA segment. The molecular weights of the HEV proteins range from 14 kD to 97 kD [Nazerian K. L., et al. Avian Dis. 35:572-578 (1991)]. The 97 kD polypeptide is the structural hexon protein, a monomer of the major outer capsid. Other structural proteins are the penton base protein, having a predicted size of about 50 kD and the fiber protein which anchors the penton base protein. This fiber protein consists of a tail and a globular head, which plays an important role in the first attachment of the virus to the cell receptor.
The virus infects turkeys and causes a disease which is characterized by depression, splenomegaly, intestinal hemorrhages and immuno-suppression [Domermuth C. H., and Gross W. B. (1991) Diseases of Poultry, 9th Edition, M. S. Hofstad et al. Eds. Iowa State University Press, Ames, Iowa]. The virus replicates in B cells and macrophages [Suresh M. and Sharma J. M. J. Virol. 70:30-36 (1996)] and is concentrated in large amounts in the spleen. Since B cells play an important role in the primary immune response, afflicted birds suffer mostly from weight loss.
Infection of birds by the HEV is especially prevalent during the ages of 7 to 9 weeks [Domermuth C. H. and Gross W. B., Diseases of Poultry, Iowa State University Press, 8th Edition pp. 511-516 (1984)]. Younger birds are protected by maternal antibodies [Van den Hurk, J. V. Avian Dis. 30:662-671 (1986); Harris J. R. and Domermuth C. H., Avian Dis. 21:120-122 (1977); Fadly, A. M. and Nazerian K. Avian Dis. 33:778-786 (1989)]. The rate of mortality of infected birds is high and, since the immune response is damaged, the surviving birds exhibit high vulnerability to other diseases. Moreover, infection with HEV reduces the effectiveness of response to various vaccines. As a result of lowered resistance, an outbreak of a HEV infection may further lead to outbreaks of other diseases. Naturally, such events result in heavy financial loss to the breeders.
Infectious diseases in animals, and in particular in farm animals, are one of the most important economic factors in agriculture, for example, in the poultry industry. The minimalization of losses from diseases, by means of effective vaccines, plays a major part in achieving profit in today""s intensive agricultural industry. The health of domesticated animals depends on management, on a proper vaccination system and on the availability of effective vaccines.
Since the price of a single farm animal is relatively low, the cost of production and delivery of the vaccine becomes critical. Naturally, if cost of the production of the vaccine is too high, its use will not be economically worthwhile.
In the last decade recombinant adenoviral vectors have become a subject for research as vectors in gene therapy [Kozarsky K. F. and Wilson J. M., Current Options in Genetics and Development 3:499-503 (1993)]. The complete sequence of the viral DNA is essential for enabling successful manipulation of the virus, for use in gene delivery. To date, recombinant Ad""s have been employed in a variety of gene therapy applications as carriers of foreign genes, as obtained with vaccinia and fowlpox [Yamanouchi K. K., et al. The Veterinary Record 13:152-156 (1993); Boursnell M. E. G. et al Virology 265:18634-18642 (1990), respectively] and in sub-unit vaccination [Israel Patent Application No.122626].
One aim of the present invention is to construct a recombinant HEV. There are a number of advantages in utilizing an adenoviridae type transfection and expression system such as the HEV derived vector of the present invention. These viruses are easy to grow giving high titers, and both the virions and the viral genome are very stable. Very high levels of expression are possible since most of the macromolecular biosynthesis in adenovirus-infected cells is virus directed at late time points post infection. Proteins made in Ad-derived expression systems would be expected to have all the post-translational modifications that might be important in determining their functional and antigenic properties, and may thus be useful for therapeutic, diagnostic or vaccination purposes.
In addition, the Ad genome is relatively easy to manipulate by recombinant DNA techniques, allowing incorporation of foreign genes as large a 7.5 kb. The ability or recombinant Ad""s to terminally transduce differentiated cells in vivo has made these vectors important candidates for many gene therapy applications [S. I., Michael et al. Gene Therapy 2:660-668 (1995)]. Evidently, knowledge of the complete sequence of the viral DNA is needed in order to perform the required manipulations in the sequence.
Although permissive infections are ultimately lytic, infected cells remain intact until relatively late in infection, making the collection of concentrated virus and virus specified intracellular proteins fairly visible.
The present invention relates to a DNA sequence which comprises (a) a nucleotide sequence substantially as shown in SEQ ID NO:1; or (b) a nucleotide sequence which corresponds to the sequence substantially as shown in SEQ ID NO:1 within the scope of the degeneracy of the genetic code; or (c) a nucleotide sequence which hybridizes, under conditions that allow such hybridization to occur, with the sequences according to (a) or (b) or with a fragment thereof.
The DNA sequence of the invention encodes a Hemorrhagic Enteritis Virus (HEV) and biologically functional homologues and fragments thereof, or a non-virulent HEV which is capable of infecting a host cell upon exposure thereto.
The invention also concerns with a DNA sequence comprising (a) at least one nucleotide sequence selected from the nucleotide sequences substantially as shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30 and functional fragments thereof, said fragment being other than the nucleotide sequences substantially as shown in SEQ ID NO""s:6 and 14 and other than the nucleotide sequence comprising nucleotide bases 187 to 1358 of the nucleotide sequence substantially as shown in SEQ ID NO:22;or (b) at least one nucleotide sequence selected from the nucleotide sequence which corresponds to the above sequences, within the scope of the degeneracy of the genetic code; or (c) a nucleotide sequence which hybridizes, under conditions that allow such hybridization to occur, with the sequence according to (a) or (b) or with a fragment thereof.
In a second aspect, the invention relates to a vector comprising the DNA sequence of the invention or deletions, insertions, mutations, replacements or modifications thereof.
In addition, the invention relates to a DNA construct comprising the DNA sequence of the invention or deletions, insertions, mutations, replacements or modifications thereof and at least one therapeutic exogenous nucleotide sequence operably linked thereto, the DNA construct being capable of transfecting a host cell, upon exposure thereto, with the exogenous nucleotide sequence, the exogenous nucleotide sequence being a sequence which is not made or contained in the cell or is made or contained in the cell in defective form.
In yet a further aspect, the invention relates to a DNA construct for the expression of a therapeutic protein or peptide product in a host cell, comprising a vector of the invention and at least one exogenous nucleotide sequence operably linked thereto which encodes the protein or peptide product.
In another aspect, the invention concerns host cells transformed with a nucleotide sequence of the invention and with host cells transfected with the DNA constructs of the invention.
In yet an additional aspect, the invention relates to a protein or peptide expressed by the host cells of the invention, wherein the protein or peptide product is a therapeutic protein or peptide product which is not made or contained in the cell, or is a therapeutic protein or peptide product which is made or contained in the cell in abnormally low amount, or is a therapeutic protein or peptide product which is made or contained in the cell in defective form or is a therapeutic protein or peptide product which is made or contained in the cell in physiologically abnormal amounts.
Alternatively, the invention relates to a protein or peptide product comprising a sequence substantially as shown in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or functional equivalents and fragments thereof, said fragment being other than the amino acid sequences substantially as shown in SEQ ID NO""s 6 and 14 and other than the amino acid sequence comprising amino acids 63 to amino acid 454 of the amino acid sequence substantially as shown in SEQ ID NO 23.
The invention also relates to a vaccine for immunizing a domesticated animal against at least one specific antigen comprising the DNA construct of the invention or the protein of the invention, optionally further comprising pharmaceutically acceptable carriers, diluents and additives.
Pharmaceutical compositions comprising as active ingredient a therapeutically effective amount of the DNA construct of the or of the cells of the invention or of the therapeutic protein or peptide product of the invention are also within the scope of the invention.
Finally, the different uses of the nucleotide sequence of the invention or the DNA constructs of the invention or the proteins of the invention in the preparation of a vaccine or a pharmaceutical composition also constitute part of the invention.